Combined use of conventional and clumped carbonate stable isotopes to identify hydrothermal isotopic alteration in cave walls

Alteration of conventional carbonate stable isotopes (δ18O, δ13C) in cave walls has been shown to be a useful tool to identify cave formation driven by deep-seated processes, i.e., hypogene karstification. If combined with a prior information on the paleowater stable isotope composition, further insights can be obtained on the temperature and the source of the paleowater. Clumped isotope composition (Δ47) of carbonates is an independent measurement of temperature, and if combined with the conventional stable isotopes, can provide information on the paleowater stable isotope composition. On the example of Provalata Cave (N. Macedonia), we apply for the first time, both conventional and clumped stable isotope analysis, and identify two different isotope alteration trends, reflecting two distinct hydrothermal events: an older, hotter one, where isotope alteration was likely related to isotope diffusion, lowering the δ18O values of the carbonate; and a younger one, related to the cave formation by low-temperature CO2-rich thermal waters, with dissolution-reprecipitation as the alteration mechanism, causing decrease in δ18O values, and unexpected increase in δ13C values. The findings are further corroborated by additional insight from optical petrography and cathodoluminescence microscopy, as well as fluid inclusion analysis of secondary calcite crystals related to the cave forming phase.


Sampling location and sample description
Samples were collected along the western and northern wall in the First Room of Provalata Cave (Figs.SS3, SS4). PR20 and PROV03 were cut from calcite coatings along the cave wall using circular saw with diamond blade. Two cores (C2 and C3) were drilled using 2.5 cm diameter corer through calcite coating and underlying marble bedrock, using the same approach as described in Spötl & Mattey (2012). Figure S3. Location of studied Provalata Cave samples: circle -core sample; square -hand sample.
PR20 is ~ 20 mm thick calcite crust cut from the northern wall of the First Room. PROV03 is ~18 cm long cut through the marble bedrock and calcite coating from the norther side of the First Room, at the junction with a small niche. C2 is a ~30 cm long core drilled at the western wall in the First Room, close to the entrance shaft. It covers ~15 cm calcite coating and ~15 cm marble bedrock. C3 core is a small ~64 mm long core drilled next to where PR20 crust was sampled. The calcite coating in the core is thin (26 mm), as it was largely dissolved in the subsequent sulfuric acid speleogenetic phase. The collected samples were cut in half and grinded. Subsamples were drilled for carbonate stable isotope analysis by a hand drill along the sample length, perpendicular to the cave wall, and following the growth axis of the calcite coatings (Fig. S5). The marble section and the first 2 cm of the calcite coating at C2 were sampled at a resolution of 1-5 mm for conventional stable isotope analysis. Additionally, C2, and the other samples were drilled at a lower resolution for combined conventional and clumped stable isotope analysis. Three solid prisms were cut from the calcite coating in C2 at two locations for U-Pb and U-series analysis (Fig.  S5).
Similarly, calcite marble bedrock samples collected along the NNW-SSE stripe (Fig.S1) were also hand-drilled for conventional and clumped stable isotope analysis.
Thin sections were prepared from samples PR20 and C2 for petrographic and fluid inclusion analysis.
For fluid inclusion stable isotope and noble gas analysis, subsamples were cut from PR20, C3 and PROV03 samples (Fig. S5). Figure S5. Studied samples from Provalata Cave and location of collected subsamples for various analyses. Figure S6. Photomicrographs of C2 core showing different sections of the calcite marble cave wall in plane-polarized transmitted light and cathodoluminescent light. (a) Overview of the thin section from the outer part of the core with location of the closer examined sections in b to g. (b) View of the unaltered section of the marble having large (mm size) crystals with dark blue luminescence. (c) A cross-section from the unaltered (left) through pale grey (middle) to white rim section, showing dark blue, violet and orange luminescence, respectively. (d) Part of the white rim, with remnants of a large calcite crystal with dark blue luminescence that changes outwards to violet, surrounded by smaller crystals with orange luminescence. (e) Outermost part of the alteration profile dominated by small crystals with orange luminescence, with larger crystals with orange luminescence visible towards the edge as part of the covering calcite coating. (f-j) Close-up view of the white altered rim section showing very small calcite crystals filling up pore spaces.

Modeling of carbon isotope composition in secondary calcite minerals
For the modeling of the δ 13 Ccc values in terms of calcite precipitation due to change in temperature with or without CO2 degassing, we have used the equation of Zheng (1990) with a modification, that instead of considering two extreme cases of HCO3-dominant or H2CO3dominant fluid, for which only fractionation of calcite-HCO3 or calcite-H2CO3 is used, respectively, we select a set of fractions of H2CO3 in the DIC of the fluid (where DIC = fH2CO3 + (1-f) × HCO3), that itself reflects fluid pH at a given T. Thus, for a selected δ 13 CDIC (-1 to +4 ‰), range of temperatures (5-40 °C) and fH2CO3 (0.3-0.7), first we calculate the δ 13 C of the CO2 in equilibrium with the fluid (δ 13 CCO2) from the relationship: and then we calculate δ 13 Ccc values using the Rayleigh model equation of Zheng (1990), in the form of: where Xc is the mole fraction of carbon in the degassed CO2, assuming that chemically the mol fraction of carbon lost due to CO2 degassing is identical to the one lost through calcite precipitation, and 1000lnαH2CO3-CO2, 1000lnαHCO3-CO2 and 1000lnαcc-CO2 are the temperature dependent carbon fractionation factors of H2CO3, HCO3 and calcite with CO2, respectively (Mook 2000).
The modeled curves show characteristic slopes of T-δ 13 Ccc for a range of fH2CO3 (Figs. S8, S9), having positive slope at low fH2CO3 (higher pH) and slightly negative slope at higher fH2CO3 (lower pH). For a given fH2CO3, the intercept is primarily controlled by the δ 13 CDIC, but also by the degassing, with higher Xc value showing lower intercept. Negative slope is maintained only for conditions of higher fH2CO3 and no or low degassing (>0.5 fH2CO3 at 0 Xc, >0.6 fH2CO3 at 0.1 Xc and >0.7 fH2CO3 at 0.2 Xc). Figure S8. Modeling of change in δ 13 Ccc with change in temperature for fH2CO3 of 0.3 to 0.7, and a δ 13 CDIC of -1‰ to +4‰ (a-f), for an Xc of 0.1 (i.e., 0.1 mole fraction of carbon in the degassed CO2). Also shown are curves for H2CO3-dominant (thick full line) and HCO3dominant fluid (thick dash-dot line), calculated using the original equation of Zheng (1990). Figure S9. Modeling of change in δ 13 Ccc with change in temperature for fH2CO3 of 0.3 to 0.7, and a δ 13 CDIC of -1‰ to +4‰ (a-f), for an Xc of 0 (no CO2 degassing). Also shown are curves for H2CO3-dominant (thick full line) and HCO3-dominant fluid (thick dash-dot line), calculated using the original equation of Zheng (1990).
To gain insight into the composition of the DIC at the onset of calcite deposition in C2 core (δ 13 Ccc value of +7.9‰ and apparent temperature of 12°C), we model the δ 13 CDIC value of a calcite saturated fluid (SIcc≥0) at a range of pH values and temperature of 12°C, so that the calcite precipitating from it under isotopic equilibrium conditions, has δ 13 Ccc value of +7.9‰. We first calculate the fractions of H2CO3 and HCO3 in PHREEQC Version 3 software (Parkhurst and Appelo 2013) for a range of pH values (6 to 7.6), with temperature set at 12°C, and calcium and DIC concentrations determined by forcing charge balance to the solution at SIcc of 0.01.
We determined the δ 13 CCO2 value for the CO2 in equilibrium with calcite with δ 13 Ccc value of +7.9‰ from: and then we calculate the δ 13 CDIC value using equation (1).
Using the PHREEQC modeled Ca 2+ and DIC concentrations, we can separate the fractions of carbon in the DIC (CDIC) derived from dissolution of CaCO3 (Crock) and from external CO2 (Cext): (4) The isotopic composition of the external CO2 i.e., CO2 not accounted for by dissolution of carbonate rocks (δ 13 Cext) can be determined from the following equation (Chiodini et al. 2000): It should be noted that the outlined approach considers only pure CaCO3 dissolution, i.e., the rock component is represented solely by the Ca 2+ ion concentration. If Mg 2+ is also introduced, e.g., by dissolution of dolomite, calcite saturation can be reached at lower Ca 2+ concentrations, but there will be no significant change in the δ 13 CDIC, and if the dolomite has similar δ 13 Crock, there will be no significant change in the δ 13 Cext as well. To demonstrate, we do the same water chemistry calculation in PHREEQC, but we include Mg 2+ concentration that is half the one of Ca 2+ calculated previously. The DIC and Ca 2+ are constrained in the same way by forcing charge balance and SIcc of 0.01. The final Mg/Ca ratio of the modeled compositions is 0.6.
Modeled values using δ 13 Crock of +2.5‰, as found for the calcite marble formation, and SIcc of 0.01 are given in Table S4 and curves are shown in Fig. S10.